CLONING AND SEQUENCING OF THE REPLICATION ORIGIN (ORIC) OF THE SPIROPLASMA-CITRI CHROMOSOME AND CONSTRUCTION OF AUTONOMOUSLY REPLICATING ARTIFICIAL PLASMIDS

A 5.6-kbp fragment of Spiroplasma citri DNA containing the dnaA gene h as been cloned and sequenced. Nucleotide sequence analysis shows that this fragment harbors the genes for the replication initiator protein (dnaA), the beta subunit of DNA polymerase III (dnaN), and the DNA gyr ase subunits A and B (gyrA and gyrB). The arrangement of these genes, dnaA-dnaN-gyrB-gyrA A, is similar to that found in all Gram-positive b acterial genomes studied so far, except that no recF gene was found be tween dnaN and gyrB. Several DnaA-box consensus sequences were found u pstream of dnaA and in the dnaA-dnaN intergenic region. The dnaA regio n with the flanking DnaA-boxes and the tetracycline resistance determi nant, tetM, were linked into a circular recombinant DNA. This DNA was able to replicate autonomously when introduced by electroporation into S. citri cells. These experiments show that the dnaA region with the DnaA-boxes is the origin of replication of S. citri and can be used to construct gene vectors.